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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-KAP1 antibody [EPR5249] (ab109545)

Reviews (1)Q&A (6)References (1)
Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Immunocytochemistry/ Immunofluorescence - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Immunoprecipitation - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Flow Cytometry (Intracellular) - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Anti-KAP1 antibody [EPR5249] (ab109545)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR5249] to KAP1
  • Suitable for: Flow Cyt (Intra), IP, WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Carrier Free

Overview

  • Product name

    Anti-KAP1 antibody [EPR5249]
    See all KAP1 primary antibodies

  • Description

    Rabbit monoclonal [EPR5249] to KAP1

  • Host species

    Rabbit

  • Specificity

    Although the immunogen is from a phosphor-peptide, this antibody detects phospho and non-phospho KAP1. Based on a peptide blocking experiment it has been found that the signal generated after non-phospho peptide blocking became much weaker, thus indicating that ab109545 shows cross-reactivity with the non-phospho KAP1 at high level.

  • Tested applications

    Suitable for: Flow Cyt (Intra), IP, WB, IHC-P, ICC/IFmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: A431 and MCF7 cell lysates; IHC-P: Human colon and kidney tissue; ICC/IF: Hela cells; IP: A431 cell lysate.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab109545 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt (Intra)
1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP
1/30.
WB (1)
1/10000 - 1/50000. Detects a band of approximately 110 kDa (predicted molecular weight: 89 kDa).
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol. Heat up to 98 degrees C, below boiling, and then let cool for 10-20 min.
ICC/IF
1/100 - 1/250.
Notes
Flow Cyt (Intra)
1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP
1/30.
WB
1/10000 - 1/50000. Detects a band of approximately 110 kDa (predicted molecular weight: 89 kDa).
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol. Heat up to 98 degrees C, below boiling, and then let cool for 10-20 min.
ICC/IF
1/100 - 1/250.

Target

  • Function

    Nuclear corepressor for KRAB domain-containing zinc finger proteins (KRAB-ZFPs). Mediates gene silencing by recruiting CHD3, a subunit of the nucleosome remodeling and deacetylation (NuRD) complex, and SETDB1 (which specifically methylates histone H3 at 'Lys-9' (H3K9me)) to the promoter regions of KRAB target genes. Enhances transcriptional repression by coordinating the increase in H3K9me, the decrease in histone H3 'Lys-9 and 'Lys-14' acetylation (H3K9ac and H3K14ac, respectively) and the disposition of HP1 proteins to silence gene expression. Recruitment of SETDB1 induces heterochromatinization. May play a role as a coactivator for CEBPB and NR3C1 in the transcriptional activation of ORM1. Also corepressor for ERBB4. Inhibits E2F1 activity by stimulating E2F1-HDAC1 complex formation and inhibiting E2F1 acetylation. May serve as a partial backup to prevent E2F1-mediated apoptosis in the absence of RB1. Important regulator of CDKN1A/p21(CIP1). Has E3 SUMO-protein ligase activity toward itself via its PHD-type zinc finger.

  • Tissue specificity

    Expressed in all tissues tested including spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes.

  • Pathway

    Protein modification; protein sumoylation.

  • Sequence similarities

    Belongs to the TRIM/RBCC family.
    Contains 2 B box-type zinc fingers.
    Contains 1 bromo domain.
    Contains 1 PHD-type zinc finger.
    Contains 1 RING-type zinc finger.

  • Domain

    The HP1 box is both necessary and sufficient for HP1 binding.
    The PHD-type zinc finger enhances CEBPB transcriptional activity. The PHD-type zinc finger, the HP1 box and the bromo domain, function together to assemble the machinery required for repression of KRAB domain-containing proteins. Acts as an intramolecular SUMO E3 ligase for autosumoylation of bromodomain.
    The RING-finger-B Box-coiled-coil/tripartite motif (RBCC/TRIM motif) is required for interaction with the KRAB domain of KRAB-zinc finger proteins. Binds four zinc ions per molecule. The RING finger and the N-terminal of the leucine zipper alpha helical coiled-coil region of RBCC are required for oligomerization.
    Contains one Pro-Xaa-Val-Xaa-Leu (PxVxL) motif, which is required for interaction with chromoshadow domains. This motif requires additional residues -7, -6, +4 and +5 of the central Val which contact the chromoshadow domain.

  • Post-translational
    modifications

    Phosphorylated upon DNA damage, probably by ATM or ATR. ATM-induced phosphorylation on Ser-824 represses sumoylation leading to the de-repression of expression of a subset of genes involved in cell cycle control and apoptosis in response to genotoxic stress. Dephosphorylation by the phosphatases, PPP1CA and PP1CB forms, allows sumoylation and expression of TRIM28 target genes.
    Sumoylation/desumoylation events regulate TRIM28-mediated transcriptional repression. Sumoylation is required for interaction with CHD3 and SETDB1 and the corepressor activity. Represses and is repressed by Ser-824 phosphorylation. Enhances the TRIM28 corepressor activity, inhibiting transcriptional activity of a number of genes including GADD45A and CDKN1A/p21. Lys-554, Lys-779 and Lys-804 are the major sites of sumoylation. In response to Dox-induced DNA damage, enhanced phosphorylation on Ser-824 prevents sumoylation and allows de-repression of CDKN1A/p21.

  • Cellular localization

    Nucleus. Associated with centromeric heterochromatin during cell differentiation through CBX1.
  • Information by UniProt

  • Database links

    • Alternative names

      • E3 SUMO protein ligase TRIM28 antibody
      • E3 SUMO-protein ligase TRIM28 antibody
      • FLJ29029 antibody
      • KAP 1 antibody
      • KAP-1 antibody
      • KRAB associated protein 1 antibody
      • KRAB interacting protein 1 antibody
      • KRAB-associated protein 1 antibody
      • KRAB-interacting protein 1 antibody
      • KRIP 1 antibody
      • KRIP-1 antibody
      • KRIP1 antibody
      • Nuclear corepressor KAP 1 antibody
      • Nuclear corepressor KAP-1 antibody
      • RING finger protein 96 antibody
      • RNF96 antibody
      • TF1B antibody
      • TIF1 beta antibody
      • TIF1-beta antibody
      • TIF1B antibody
      • TIF1B_HUMAN antibody
      • Transcription intermediary factor 1 beta antibody
      • Transcription intermediary factor 1-beta antibody
      • Trim28 antibody
      • Tripartite motif containing 28 antibody
      • tripartite motif containing protein 28 antibody
      • Tripartite motif-containing protein 28 antibody
      see all

    Images

    • Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
      Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)

      Lane 1: Wild type HAP1 whole cell lysate (20 µg)
      Lane 2: HAP1 + DMSO whole cell lysate (20 µg)
      Lane 3: HAP1 + Blaomycin whole cell lysate (20 µg)
      Lane 4: TRIM28 knockout HAP1 whole cell lysate (20 µg)
      Lane 5: TRIM28 knockout HAP1 + DMSO whole cell lysate (20 µg)
      Lane 6: TRIM28 knockout HAP1 + Blaomycin whole cell lysate (20 µg)
      Lane 7: HeLa + DMSO whole cell lysate (20 µg)
      Lane 8: HeLa + Blaomycin whole cell lysate (20 µg)

      Lanes 1 - 8: Merged signal (red and green). Green - ab109545 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.

      ab109545 was shown to specifically react with KAP1 in wild type cells as signal was lost in KAP1 knockout cells. Wild-type and KAP1 knockout samples were subjected to SDS-PAGE. ab109545 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

    • Immunocytochemistry/ Immunofluorescence - Anti-KAP1 antibody [EPR5249] (ab109545)
      Immunocytochemistry/ Immunofluorescence - Anti-KAP1 antibody [EPR5249] (ab109545)
      ab109545, at a 1/100 dilution, staining KAP1 in HeLa cells
    • Immunoprecipitation - Anti-KAP1 antibody [EPR5249] (ab109545)
      Immunoprecipitation - Anti-KAP1 antibody [EPR5249] (ab109545)
      KAP1 was immunoprecipitated from 0.35 mg A431 (Human epidermoid carcinoma epithelial cell) cell lysate 10 µg with ab109545 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab109545 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

      Lane 1: A431 (Human epidermoid carcinoma epithelial cell) cell lysate 10 µg

      Lane 2: ab109545 IP in A431 cell lysate

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab109545 in A431 cell lysate

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 32 seconds

    • Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
      Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
      All lanes : Anti-KAP1 antibody [EPR5249] (ab109545) at 1/10000 dilution

      Lane 1 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates
      Lane 2 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates

      Lysates/proteins at 15 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

      Developed using the ECL technique.

      Predicted band size: 89 kDa
      Observed band size: 110 kDa why is the actual band size different from the predicted?


      Exposure time: 10 seconds


      Blocking/diluting buffer and concentration: 5% NFDM/TBST

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)

      ab109545, at a 1/100 dilution, staining KAP1 in formalin-fixed, paraffin-embedded Human colon tissue.

      Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)

      ab109545, at a 1/100 dilution, staining KAP1 in formalin-fixed, paraffin-embedded Human kidney tissue.

      Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

    • Flow Cytometry (Intracellular) - Anti-KAP1 antibody [EPR5249] (ab109545)
      Flow Cytometry (Intracellular) - Anti-KAP1 antibody [EPR5249] (ab109545)

      Overlay histogram showing HeLa cells stained with ab109545 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab190545, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

       

    • Anti-KAP1 antibody [EPR5249] (ab109545)
      Anti-KAP1 antibody [EPR5249] (ab109545)

    References (1)

    Publishing research using ab109545? Please let us know so that we can cite the reference in this datasheet.

    ab109545 has been referenced in 1 publication.

    • Peng Y  et al. TRIM28 activates autophagy and promotes cell proliferation in glioblastoma. Onco Targets Ther 12:397-404 (2019). PubMed: 30655676

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-7 of 7 Abreviews or Q&A

    Western blot abreview for Anti-KAP1 (phospho S473) antibody [EPR5249]

     Below Average
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Hela cells)
    Loading amount
    50 µg
    Specification
    Hela cells
    Treatment
    non treated, AZD7762 for 4 hrs, etoposide 2µM for 3 hrs, 50 nM AZD7762 for 1h+ etoposide 3h
    Gel Running Conditions
    Reduced Denaturing (8%)
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 18°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted May 15 2012

    Question

    What is the concentration of lot GR564755-8 of ab109545?

    Read More
    Answer

    The IgG concentration of lot GR564755 (including GR564755-8) is 0.166 mg/ml.

    Read More

    Question



    Inquiry: I am running Western blots with rat brain samples using ZFP47 antibody (ab45341), as well as KAP1 antibody (ab22553) and pS473-KAP1 antibody (ab109545). I wonder whether these antibodies have been tested for their specificity (e.g. using knockout/knockdown tissues, using tissues known to not express these proteins). Thank you!

    Read More
    Answer

    Thank you for contacting us. No, these antibodies have not been tested with overexpression or knockdown cells.


    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

    Question

    Dear Sir

    Any news on the anti-KAP1-phospho S473 antibody?

    Sincerely

    Read More
    Answer

    Thank you for your kind reminder.

    As I have mentioned in my previous e-mail (sent Thu 21 Jun 12 ·11:32) we could offer some alternative antibodies for you. If you can use phospho specific anti KAP1 antibody other than S473 then we can certainly offer any of these to try ab133225 or ab70369 or ab84077.

    If you wish otherwise, I could arrange a full refund for you.

    Could you please get back to me and let me know how you wish to proceed.

    Read More

    Question

    We normally block PVDF membranes with non-fat milk, but the incubation with the phosphospecific antibody is done in 5% BSA (as written below). This protocol has consistently worked for all phosphospecific antibodies we have used so far (see our publications).

    On a few occasions in the past we tried blocking membranes with BSA, but the background was unacceptably high with phosphospecific antibodies.

    My impression with your anti-KAP1-pS473 antibody is that it hasn’t been properly purified, as it reacts with total KAP1 but not with pS473.

    Best regards

    Read More
    Answer

    Thank you for your response and patience.

    I have been discussing this enquiry with my colleagues in the lab. Could you please confirm if you have tried our positive control treatment, Lambda Phosphatase?

    I understand that radiation also works based on the literature. I attached a dot blot to show phospho specificity. “N-P” stands for the control peptide and “P” for the phospho peptide. Please let me know if you have any questions.

    I hope this helps and if I can assist further, please do not hesitate to contact me.

    Read More

    Question

    We have recently bought (through the local distributor) the Anti-KAP1 (phospho S473) antibody ab109545 LOT: GR54755-3 that when tested on western blots of lysates from human cells treated or untreated with radiation shows a band of the correct MW but whose levels do not change with treatment (see attached). We believe that the antibody in NOT recognizing S473 phosphorylation, but only total KAP1.
    According to previously published data, KAP1 S473 phosphorylation occurs only after treatment of cells with genotoxic stress, such as ionizing radiation ( see http://www.ncbi.nlm.nih.gov/pubmed/22491012 Lee DH, et al, EMBO J. 2012 Apr 10. doi: 10.1038/emboj.2012.86. )
    I would appreciate if you could send a replacement antibody of a different lot and after you checked it works as expected.

    Please find attached the completed form.

    I draw your attention on our longstanding technical expertise in the analysis of phosphorylated proteins from DNA damage cells (see papers listed below).


    1) Abcam product code ab: 109545

    2) Abcam order reference number or product batch number: GR54755-3

    3) Description of the problem:

    Antibody not recognizing KAP1- S473 phosphorylation after radiation treatment

    4) Sample preparation:

    Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysates from MCF7 cells

    Lysis buffer: 4% SDS + 125mM Tris HCl

    Protease inhibitors: PMSF

    Phosphatase inhibitors: Na3Vo4

    Reducing agent: β-mercaptoethanol

    Boiling for ≥5 min? yes/no: Yes

    Protein loaded ug/lane or cells/lane: 40µg/lane

    Positive control: irradiated MCF7 cells

    Negative control: untreated shCHK2 MCF7 cells

    5) Percentage of gel: 4-12 % Bis-Tris gel from Invitrogen

    Type of membrane: PVDF

    Protein transfer verified: YES

    Blocking agent and concentration: 4% Milk in 1XPBS+Tween(0.1%)

    Blocking time: 1 hour

    Blocking temperature: Room Temperature

    6) Primary antibody (If more than one was used, describe ifn “additional notes”) :

    Concentration or dilution: 1:50000

    Diluent buffer : 5% BSA in TBS+Tween 0.1%

    Incubation time: overnight

    Incubation temperature: 15ºC

    7) Secondary antibody: Donkey Anti-Rabbit IgG ECL antibody

    Species Donkey

    Reacts against: Rabbit

    Concentration or dilution: 1:2000

    Diluent buffer : 4% Milk in 1XPBS+Tween 0.1%

    Incubation time: 1 Hour

    Incubation temperature: 15ºC

    Fluorochrome or enzyme conjugate: HRP

    8) Washing after primary and secondary antibodies Buffer: 1XPBS Tween (0.1%)

    Number of washes: 3 washes, 10 minutes each

    9) Detection method: ECL

    10) How many times have you run this staining? 3 Times

    Do you obtain the same results every time? YES

    What steps have you altered to try and optimize the use of this antibody? Cell lines, antibody dilution and treatments with different DNA damaging agents

    Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.

    Best regards

    Read More
    Answer

    Thank you for your enquiry regarding ab109545 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

    I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

    The protocol looks fine to me. There is only one thing I would like to comment on is the blocking:

    For detection of phospho protein, we would suggest using 3% BSA solution - as blocking agent - rather than milk. Milk may lead to significant decrease or loss of signal intensity due to reaction of primary antibody with blocking/diluting agent.

    Have you used this antibody before? Could you please confirm it?

    Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

    Read More

    Question

    Dear Sir

    We have recently bought (through the local distributor) the Anti-KAP1 (phospho S473) antibody ab109545 LOT: GR54755-3 that when tested on western blots of lysates from human cells treated or untreated with radiation shows a band of the correct MW but whose levels do not change with treatment (see attached). We believe that the antibody in NOT recognizing S473 phosphorylation, but only total KAP1.

    According to previously published data, KAP1 S473 phosphorylation occurs only after treatment of cells with genotoxic stress, such as ionizing radiation ( see http://www.ncbi.nlm.nih.gov/pubmed/22491012 Lee DH, et al, EMBO J. 2012 Apr 10. doi: 10.1038/emboj.2012.86. )

    I would appreciate if you could send a replacement antibody of a different lot and after you checked it works as expected.

    Yours sincerely

    Read More
    Answer

    Thank you for your enquiry regarding ab109545 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody and do not get the expected results.

    I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

    Could you provide some further details of the protocol used and complete the following form (attached as a word document).

    I am particularly interested in the followings:

    - Sample preparation:

    - Lysis buffer:

    - Blocking agent used:

    Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

    Read More

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